Ariel A. Petruk

Protein dynamics and ligand migration interplay as studied by computer simulation.

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: Computer simulation studies in model tyrosine–cysteine peptides in solution

Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O(·)) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O(·) to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr-Cys residues.

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER*

Background: Superoxide dismutases are inactivated by peroxynitrite. Results: T. cruzi cytosolic Fe-SODB is highly resistant toward peroxynitrite-mediated tyrosine nitration and inactivation as compared with mitochondrial Fe-SODA. Conclusion: Intramolecular electron transfer in Fe-SODB from Cys83 to critical Tyr35 prevents enzyme nitration and inactivation. Significance: Disparate susceptibilities of Fe-SODs to peroxynitrite can influence parasite virulence during T. cruzi infection of mammalian cells. Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 104 m−1 s−1 and 4.3 ± 0.4 × 104 m−1 s−1 at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr35. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys83 mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys83 present in Fe-SODB acts as an electron donor that repairs Tyr35 radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.

Solvent structure improves docking prediction in Lectin- Carbohydrate complexes

Recognition and complex formation between proteins and carbohydrates is a key issue in many important biological processes. Determination of the three-dimensional structure of such complexes is thus most relevant, but particularly challenging because of their usually low binding affinity. In silico docking methods have a long-standing tradition in predicting protein-ligand complexes, and allow a potentially fast exploration of a number of possible protein-carbohydrate complex structures. However, determining which of these predicted complexes represents the correct structure is not always straightforward. In this work, we present a modification of the scoring function provided by AutoDock4, a widely used docking software, on the basis of analysis of the solvent structure adjacent to the protein surface, as derived from molecular dynamics simulations, that allows the definition and characterization of regions with higher water occupancy than the bulk solvent, called water sites. They mimic the interaction held between the carbohydrate -OH groups and the protein. We used this information for an improved docking method in relation to its capacity to correctly predict the protein-carbohydrate complexes for a number of tested proteins, whose ligands range in size from mono- to tetrasaccharide. Our results show that the presented method significantly improves the docking predictions. The resulting solvent-structure-biased docking protocol, therefore, appears as a powerful tool for the design and optimization of development of glycomimetic drugs, while providing new insights into protein-carbohydrate interactions. Moreover, the achieved improvement also underscores the relevance of the solvent structure to the protein carbohydrate recognition process.

Interaction between proteins and Ir based CO releasing molecules: mechanism of adduct formation and CO release.

Carbon monoxide releasing molecules (CORMs) have important bactericidal, anti-inflammatory, neuroprotective, and antiapoptotic effects and can be used as tools for CO physiology experiments, including studies on vasodilation. In this context, a new class of CO releasing molecules, based on pentachlorocarbonyliridate(III) derivative have been recently reported. Although there is a growing interest in the characterization of protein-CORMs interactions, only limited structural information on CORM binding to protein and CO release has been available to date. Here, we report six different crystal structures describing events ranging from CORM entrance into the protein crystal up to the CO release and a biophysical characterization by isothermal titration calorimetry, Raman microspectroscopy, and molecular dynamics simulations of the complex between a pentachlorocarbonyliridate(III) derivative and hen egg white lysozyme, a model protein. Altogether, the data indicate the formation of a complex in which the ligand can bind to different sites of the protein surface and provide clues on the mechanism of adduct formation and CO release.

QM/MM study of the C—C coupling reaction mechanism of CYP121, an essential Cytochrome p450 of Mycobacterium tuberculosis

Among 20 p450s of Mycobacterium tuberculosis (Mt), CYP121 has received an outstanding interest, not only due to its essentiality for bacterial viability but also because it catalyzes an unusual carbon–carbon coupling reaction. Based on the structure of the substrate bound enzyme, several reaction mechanisms were proposed involving first Tyr radical formation, second Tyr radical formation, and C—C coupling. Key and unknown features, being the nature of the species that generate the first and second radicals, and the role played by the protein scaffold each step. In the present work we have used classical and quantum based computer simulation methods to study in detail its reaction mechanism. Our results show that substrate binding promotes formation of the initial oxy complex, Compound I is the responsible for first Tyr radical formation, and that the second Tyr radical is formed subsequently, through a PCET reaction, promoted by the presence of key residue Arg386. The final C—C coupling reaction possibly occurs in bulk solution, thus yielding the product in one oxygen reduction cycle. Our results thus contribute to a better comprehension of MtCYP121 reaction mechanism, with direct implications for inhibitor design, and also contribute to our general understanding of these type of enzymes. Proteins 2014; 82:1004–1021.

Mechanistic Insight into the Enzymatic Reduction of Truncated Hemoglobin N of Mycobacterium tuberculosis ROLE OF THE CD LOOP AND PRE-A MOTIF IN ELECTRON CYCLING

Background: The HbN of Mycobacterium tuberculosis carries a potent nitric-oxide dioxygenase activity despite lacking a reductase domain. Results: The NADH-ferredoxin reductase system acts as an efficient partner for the reduction of HbN. Conclusion: The interactions of HbN with the reductase are modulated by its CD loop and the Pre-A region. Significance: The present study provides new insights into the mechanism of electron transfer during nitric oxide detoxification by HbN. Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 μm/min−1, which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly48 may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10−5 eV, which is lower than the value obtained for E. coli flavoHb (8.0 10−4 eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function.

Molecular Dynamics Simulations Provide Atomistic Insight into Hydrogen Exchange Mass Spectrometry Experiments

It is now clear that proteins are flexible entities that in solution switch between conformations to achieve their function. Hydrogen/Deuterium Exchange Mass Spectrometry (HX/MS) is an invaluable tool to understand dynamic changes in proteins modulated by cofactor binding, post-transductional modifications, or protein-protein interactions. ERK2MAPK, a protein involved in highly conserved signal transduction pathways of paramount importance for normal cellular function, has been extensively studied by HX/MS. Experiments of the ERK2MAPK in the inactive and active states (in the presence or absence of bound ATP) have provided valuable information on the plasticity of the MAPK domain. However, interpretation of the HX/MS data is difficult, and changes are mostly explained in relation to available X-ray structures, precluding a complete atomic picture of protein dynamics. In the present work, we have used all atom Molecular Dynamics simulations (MD) to provide a theoretical framework for the interpretation of HX/MS data. Our results show that detailed analysis of protein-solvent interaction along the MD simulations allows (i) prediction of the number of protons exchanged for each peptide in the HX/MS experiments, (ii) rationalization of the experimentally observed changes in exchange rates in different protein conditions at the residue level, and (iii) that at least for ERK2MAPK, most of the functionally observed differences in protein dynamics are related to what can be considered the native state conformational ensemble. In summary, the combination of HX/MS experiments with all atom MD simulations emerges as a powerful approach to study protein native state dynamics with atomic resolution.

Molecular Mechanism of Myoglobin Autoxidation: Insights from Computer Simulations

Myoglobin (Mb) and hemoglobin have the biological ability to carry/store oxygen (O2), a property which requires its heme iron atom to be in the ferrous--Fe(II)--state. However, the thermodynamically stable state in the presence of O2 is Fe(III) and thus the oxidation rate of a globin is a critical parameter related to its function. Mb has been extensively studied and many mutants have been characterized regarding its oxygen mediated oxidation (i.e., autoxidation) rates. Site directed mutants in residues 29 (B10), which shapes the distal cavity, and 64 (E7), the well-known histidine gate, have been shown to display a wide range of autoxidation rate constants. In this work, we have thoroughly studied the mechanism underlying the autoxidation process by means of state-of-the-art computer simulation methodologies, using Mb and site directed mutants as benchmark cases. Our results explain the observed autoxidation rate tendencies in different variants of Mb, L29F < wt < L29A = H64Q < H64F < H64A, and shed light on several aspects of the reaction at the atomic level. First, water access to the distal pocket is a key event and the observed acid catalysis relies on HisE7 protonation and opening of the His gate to allow water access, rather than protonation of the oxy heme itself. Our results also suggest that the basic mechanism, i.e., superoxide displacement by hydroxide anion, is energetically more feasible. Finally, we confirmed that distal hydrogen bonds protect the oxy complex from autoxidation.

WATCLUST: a tool for improving the design of drugs based on protein-water interactions

MOTIVATION Water molecules are key players for protein folding and function. On the protein surface, water is not placed randomly, but display instead a particular structure evidenced by the presence of specific water sites (WS). These WS can be derived and characterized using explicit water Molecular Dynamics simulations, providing useful information for ligand binding prediction and design. Here we present WATCLUST, a WS determination and analysis tool running on the VMD platform. The tool also allows direct transfer of the WS information to Autodock program to perform biased docking. AVAILABILITY AND IMPLEMENTATION The WATCLUST plugin and documentation are freely available at http://sbg.qb.fcen.uba.ar/watclust/. CONTACT marcelo@qi.fcen.uba.ar, adrian@qi.fcen.uba.ar.